PB1910: TELAGLENASTAT (CB-839) IN MONOTHERAPY AND IN COMBINATION WITH EPIGENETIC MODULATORS OR IBRUTINIB INDUCES CELL DEATH IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS
Authors: Joana Jorge, Hugo Santiago, Beatriz Constantino, Maria Inês Costa, Beatriz Lapa, Raquel Alves, Ana Cristina Gonçalves, Ana Bela Sarmento-Ribeiro
Journal: HemaSphere (2023)

Topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research Background: Chronic lymphocytic leukemia is a lymphoproliferative disorder of clonal B lymphocytes and the most common leukemia in western countries. Glutaminolysis is a metabolic pathway that is augmented in multiple tumors, since it allows the synthesis de novo of crucial molecules for tumor progression. Glutamine has demonstrated a role in the formation of 2-hydroxiglutarate, in cells overexpressing glutaminase. 2-HG is an oncometabolite that inhibits dioxygenases that mediate epigenetic events, including DNA demethylation and histone acetylation. Telaglenastat (CB-839) is a small-molecule glutaminase inhibitor (GLS1), currently being studied in several clinical trials for the treatment of various neoplasms. Glutaminolysis and epigenetic alterations are involved in the pathophysiology of CLL and may constitute new therapeutic targets in this type of leukemia. Aims: This work investigated the therapeutic potential of Telaglenastat (CB-839), a glutaminase inhibitor, in a CLL cell line, in monotherapy and in therapeutic association with epigenetic modulators - the hypomethylating agents, azacytidine (AZA) and decitabine (DAC), and the histone deacetylates inhibitors, vorinostat (SAHA), and panobinostat (PANO) and with the BTK inhibitor, ibrutinib (IBRU). Methods: To achieve these goals, a cell model of CLL - the HG-3 cell line - was used and the effect of CB-839, in monotherapy and in therapeutic combination with epigenetic modulators and ibrutinib (2.5μM AZA, 0.5μM DAC, 0.5μM SAHA, 5nM PANO or 0.25μM IBRU) on the metabolic activity of cells was evaluated by the resazurin assay. Cell death and cell cycle were assessed by flow cytometry (FC), using double labeling with annexin V/7-AAD and PI/RNAse, respectively. Cell death was further analyzed by observing the morphological characteristics by light microscopy, after May-Grunwald Giemsa staining. Mitochondrial membrane potential was also assessed by FC, after labeling with the JC-1 probe. The expression of genes related to glutaminolysis (GLS1, GLS2, GLUD1, GLUD2) was also evaluated by qPCR. The results were statistically analyzed considering a significance level of 95% (p <0.05). Results: CB-839 reduced the metabolic activity of HG3 cells in a dose and time-dependent manner (IC25: 5nM, IC50: 25nM, 48h). Combinations of this glutaminase inhibitor with epigenetic modulators also reduced the metabolic activity (p<0.001), being the synergistic effect of the drug combination more significant with hypomethylating agents (AZA and DAC) and with IBRU. CB-839 induced cytotoxic and cytostatic effects, simultaneously by induced apoptosis and cell cycle arrest in the G0/G1 phase. In cells treated with the therapeutic combinations tested, cell death was observed predominantly by apoptosis. The association with DAC, PANO and IBRU also induced cell cycle arrest in G0/G1 phase. The drugs also induced mitochondria depolarization, with a decrease in the aggregates/monomers (A/M) ratio of JC1, suggesting that the mitochondrial pathway may be involved in the observed cell death. In cells treated with CB-839 we didn´t find noticeable changes in gene expression, with the exception of a significant increase in the expression of the GLS2 and GLUD2 genes. Summary/Conclusion CB-839 potentiated the therapeutic effect of epigenetic modulators and BTKi, suggesting a complex relationship between metabolism and epigenetic changes. In summary, the combination of glutaminolysis inhibitors and epigenetic modulators may constitute a future therapeutic alternative in CLL. Keywords: Targeted therapy, Chronic lymphocytic leukemia, Apoptosis, Epigenetic